Transcriptomics of the Vaccine Immune Response: Priming With Adjuvant Modulates Recall Innate Responses After Boosting

Abstract

Transcriptomic profiling of the immune response induced by vaccine adjuvants is of critical importance for the rational design of vaccination strategies. In this study, transcriptomics was employed to profile the effect of the vaccine adjuvant used for priming on the immune response following re-exposure to the vaccine antigen alone. Mice were primed with the chimeric vaccine antigen H56 of Mycobacterium tuberculosis administered alone or with the CAF01 adjuvant and boosted with the antigen alone. mRNA sequencing was performed on blood samples collected 1, 2, and 7 days after priming and after boosting. Gene expression analysis at day 2 after priming showed that the CAF01 adjuvanted vaccine induced a stronger upregulation of the innate immunity modules compared with the unadjuvanted formulation. The immunostimulant effect of the CAF01 adjuvant, used in the primary immunization, was clearly seen after a booster immunization with a low dose of antigen alone. One day after boost, we observed a strong upregulation of multiple genes in blood of mice primed with H56 + CAF01 compared with mice primed with the H56 alone. In particular, blood transcription modules related to innate immune response, such as monocyte and neutrophil recruitment, activation of antigen-presenting cells, and interferon response were activated. Seven days after boost, differential expression of innate response genes faded while a moderate differential expression of T cell activation modules was appreciable. Indeed, immunological analysis showed a higher frequency of H56-specific CD4+ T cells and germinal center B cells in draining lymph nodes, a strong H56-specific humoral response and a higher frequency of antibody-secreting cells in spleen of mice primed with H56 + CAF01. Taken together, these data indicate that the adjuvant used for priming strongly reprograms the immune response that, upon boosting, results in a stronger recall innate response essential for shaping the downstream adaptive response.

Keywords: CAF01; RNA sequencing; adaptive immunity; recall innate response; vaccine adjuvant.

Figure 1

Study design and samples collection. C57BL/6 mice were subcutaneously primed with H56 antigen alone or combined with CAF01, and boosted at day 28 with H56. Blood samples were collected at days 0, 14, 28, 38, 56, and 77 for antibody analysis (Ab) and at days 0, 1, 2, 7, 29, 30, and 35 (corresponding to days 1, 2, and 7 after boosting) for gene expression (Gene Exp). dLN and spleens were collected at days 0 and 38 (10 days after boosting) to evaluate the secondary CD4+ T and B cell responses.

Figure 2

Adaptive immune response following booster immunization. C57BL/6 mice were subcutaneously immunized as reported in Figure 1. dLN were collected 10 days after booster immunization and analyzed for the T and B recall responses. (A) Number of Ag-specific CD4+ T cells, identified as CD4+ CD44+ tetramer Ag85B-specific cells. Mean values ± SEM of five mice per group are reported. (B) Number of germinal center B cells, identified as GL7+ CD95+ among B220+ B cells in dLN. (C) H56-specific serum IgG titers determined on days 0, 14, 28, 38, 56, and 77 following priming, by ELISA. Antibody titers values are reported as GMT ± 95% CI of five mice per group. Arrow indicates day 28 that is the time of boosting. (D) H56-specific antibody-secreting cells (ASC) detected in spleens by ELISPOT assay. Kruskal–Wallis test was used to assess statistical differences between groups of mice primed with H56 + CAF01 versus H56 alone (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

Figure 3

Comparisons of gene expression changes elicited by H56 and H56 + CAF01 vaccine formulations. Each bivariate plot illustrates the comparison at one time point, as indicated. For the post-prime time points, the comparisons are represented relative to the naïve baseline. For post-boost time points, the baseline is the pre-boost time point. Axes represent log 2 of baseline-normalized expression values. Each dot represents a gene, and colored lines indicate the density of dots on the graph. Only genes significantly regulated (FDR < 0.05) by at least one vaccine were included and plotted. Numbers indicate the number of genes included in areas indicated by solid black lines. Dashed red lines indicate 0 values (no change in gene expression). Functional analysis was done using Reactome database, and representative significantly enriched pathways are included in captions under each plot.

Figure 4

Activation of blood transcription modules (BTMs) by the CAF01 adjuvant. Each column represents a pairwise comparison between blood RNA samples from mice primed with H56 + CAF01 versus H56 alone. Both groups were boosted after 4 weeks with H56 alone. Blood samples were collected 1, 2, and 7 days after priming and after boosting. Activation of modules was tested using the FDR-ranked lists of genes generated by edgeR generalized linear model fitting and applying the CERNO test. Rows indicate different BTMs, which were significantly (FDR < 0.001) activated in at least one time point. Each module is represented by a pie in which the proportion of significantly upregulated and downregulated genes is shown in red and blue, respectively. The gray portion of the pie represents genes that are not significantly differentially regulated. The significance of module activation is proportional to the intensity of the pie, while the effect size (area under the curve) is proportional to its size.